Clostridium difficile, a gram positive bacteria, is the primary cause of antibiotic associated pseudomembraneous colitis (PMC) in humans. The bacterium produces two toxins, toxin A a hemorrhagic enterotoxin, and toxin B a potent cytotoxin; both toxins have been reported to act synergistically to cause PMC. Outbreaks of PMC due to nosocomia spread of C. difficile have been reported in hospitals as well as day care facilities. Although the toxin genes have been sequenced, little is known about the regulation of toxin production in C. difficile. The long range objective of the proposed research is to clone and sequence the 5/DNA region upstream from the toxin B gene and to measure the amount of heterogeneity between highly toxic and low cytotoxic strains of C. difficile. To accomplish this goal a number of experiments are proposed. Regions upstream from the toxin B structural gene will be cloned into a vector and sequenced. Sequence information obtained will be used to design PCR primers for DNA amplification of 400 bp segments of the 5' region of DNA. Amplified DNA from the control line will be compared to lines with high or low cytotoxic activity using heteroduplex or SSCP analysis. Regions found to differ from the control and to correlate with cytotoxic activity will be sequenced. Heteroduplex or SSCP analysis will also be conducted on selected regions within the toxin B gene. Information obtained from the experiments should significantly contribute to information concerning the toxin genes of C. difficile.